ABSTRACT: Nitric oxide (NO) is a small, reactive molecule involved in numerous signaling pathways, including those regulating angiogenesis and metastasis. The primary cellular receptor for NO is soluble Guanylyl Cyclase (sGC), a heterodimeric hemoprotein of 150 kDa and an attractive target for the treatment of disease, including cancer. Binding of NO stimulates sGC activity, leading to the establishment of a cGMP signaling cascade. sGC is allosterically regulated by a variety of molecules, including NO, ATP, YC-1 (a small nucleotide-like pharmacophore), and by posttranslational modifications. Despite extensive study, little is known about the overall shape of sGC, the means by which allosteric regulation takes place, the arrangement of functional domains in the protein or the arrangement of the protein in the cell. We intend to fill this gap through fluorescence-based approaches that will allow us to measure structural changes within sGC, and also to monitor sGC localization within the cell. Specifically, we intend to incorporate paired fluorophores to full-length and truncated forms of sGC such that FRET measurements will reveal the distances between functional domains under stimulating and inhibiting conditions. We have developed a robust model system involving sGC from the hawkmoth {Manduca sexta) with which to begin these studies, but will also include human sGC once the fluorescence system is established. We will investigate sGC conformational states not only with isolated material, but also in live cells. Additionally, we will use a combination of immunohistology and fluorescence microscopy to monitor localization of sGC under activating and inhibiting conditions. We have shown that sGC displays a punctate arrangement in the cell, but the functional consequences of this stark pattern are unknown. Together, we expect that these studies will uncover the structural transitions that sGC undergoes and provide the framework for novel strategies in drug discovery. The generated results from this pilot project will provide the basis for future collaborative funding and research efforts.